OCR Specification focus:
‘Explain isolation of target genes, use of vectors, and formation of recombinant DNA using restriction enzymes, plasmids and DNA ligase, including electroporation.’
Genetic engineering involves deliberately modifying an organism’s DNA to introduce new traits or capabilities. It underpins biotechnology, agriculture, medicine, and synthetic biology, enabling targeted gene manipulation.
Principles of Genetic Engineering
Genetic engineering centres on the identification, isolation, modification, and transfer of genes between organisms to express desired characteristics. It relies on precise molecular techniques that allow DNA to be cut, joined, and inserted into new hosts.
The Genetic Engineering Process
The fundamental stages are:
Isolation of the desired gene from donor DNA.
Insertion of the gene into a suitable vector.
Transfer of the recombinant DNA into a host organism.
Selection and expression of the introduced gene.
Each stage requires specific enzymes and molecular tools to ensure the gene is correctly integrated and functional.
Isolation of Target Genes
The first step involves obtaining the gene coding for the desired protein. Several methods are available, each suited to different contexts.
Methods of Gene Isolation
Using restriction endonucleases: These enzymes cut DNA at specific sequences known as recognition sites, producing fragments that include the target gene.
Reverse transcription: If the gene is expressed in cells, mRNA can be used as a template to synthesise complementary DNA (cDNA) via the enzyme reverse transcriptase.
Polymerase Chain Reaction (PCR): A specific gene can be amplified directly using primers that flank the target sequence.
Restriction endonuclease: An enzyme that recognises specific short DNA sequences and cuts both strands at or near these sites, producing sticky or blunt ends.
Once isolated, the gene must be accurately inserted into a vector for transfer to the host organism.
Vectors in Genetic Engineering
A vector is a carrier DNA molecule used to deliver the target gene into a host cell. The most common vectors are plasmids and bacteriophages.
Plasmids
Plasmids are small, circular DNA molecules found naturally in bacteria. They replicate independently of chromosomal DNA, making them ideal for genetic manipulation.
Key features of plasmid vectors include:
Origin of replication (ori): Ensures the plasmid can replicate inside the host.
Selectable marker genes: Typically antibiotic resistance genes used to identify cells that have successfully taken up the plasmid.
Multiple cloning site (MCS): A region containing several restriction enzyme sites for gene insertion.
Circular map of the pBR322 cloning vector showing the origin of replication (ori), ampR and tetR selectable markers, and common restriction sites used for gene insertion. This exemplifies how plasmid vectors are annotated for cloning work. Minor extra details (specific cut sites) extend beyond the syllabus but reinforce where inserts are made. Source.
Plasmid: A small, circular DNA molecule independent of chromosomal DNA, used as a vector to carry foreign genes into host cells.
Bacteriophages
Bacteriophage vectors are viruses that infect bacteria and can be engineered to carry foreign DNA into bacterial hosts, often used when larger DNA fragments need to be transferred.
Formation of Recombinant DNA
The next stage involves combining the isolated gene with the vector DNA to create recombinant DNA.
Restriction Enzymes and Sticky Ends
Restriction endonucleases cut both the vector and the target gene at matching recognition sites, creating complementary sticky ends that facilitate joining.
Sticky ends have short overhanging sequences that base pair with complementary sequences on the insert DNA.
When matching ends are aligned, they can be permanently sealed by DNA ligase.
DNA ligase: An enzyme that catalyses the formation of phosphodiester bonds between adjacent DNA fragments, joining the sugar-phosphate backbones to form continuous strands.
The result is a stable molecule of recombinant DNA ready for introduction into a host cell.
Introduction of Recombinant DNA into Host Cells
The recombinant plasmid or vector must now enter a host organism where the gene can be expressed. Several techniques enable DNA uptake:
Transformation Methods
Electroporation: A high-voltage electric pulse temporarily disrupts the cell membrane, creating pores that allow DNA to enter.
Three-panel schematic of electroporation, showing intact membrane, transient pores forming during the pulse, and resealing afterwards as plasmid DNA enters the cell. Labels appear in Catalan, but the visual sequence and arrows clearly communicate the process. The language is an extra detail that does not affect the biological accuracy. Source.
Heat shock: Bacterial cells are exposed to calcium ions and heat to increase membrane permeability.
Microinjection: DNA is directly injected into the nucleus of a target cell using a fine glass needle.
Transduction: In bacteriophages, the virus infects the host and introduces recombinant DNA as part of its replication cycle.
Electroporation: A method of introducing DNA into cells by applying an electric field that temporarily opens pores in the cell membrane.
After DNA uptake, not all cells will successfully incorporate the recombinant molecule, so selection is essential.
Selection and Identification of Transformed Cells
To identify cells that have successfully taken up recombinant DNA, marker genes and reporter genes are employed.
Antibiotic resistance markers: Cells containing the plasmid can grow on media containing the corresponding antibiotic.
Reporter genes: Such as GFP (green fluorescent protein), produce a detectable colour or fluorescence under certain conditions.
Replica plating: A method used to identify colonies containing recombinant plasmids.
Cells with recombinant DNA are then cultured and induced to express the introduced gene, producing the desired protein or trait.
Applications of Recombinant DNA
Once integrated, the gene can be expressed, cloned, or used for further genetic modification. Key applications include:
Pharmaceutical production: e.g., recombinant insulin or vaccines.
Agricultural biotechnology: Creation of genetically modified crops with pest or herbicide resistance.
Gene research: Understanding gene function through expression in model organisms.
Industrial biotechnology: Enzyme production for detergents and biofuels.
These advances rely fundamentally on the principles of gene isolation, vector use, recombinant DNA formation, and transformation, which together form the foundation of genetic engineering.
FAQ
The type of ends depends on how the restriction enzyme cuts the DNA strands.
Sticky ends form when the enzyme cuts the two strands at different positions within the recognition site, leaving short, single-stranded overhangs that can base pair with complementary sequences.
Blunt ends form when both strands are cut at the same position, producing fragments with no overhangs.
Sticky ends are usually preferred in genetic engineering because they increase the likelihood of correct gene insertion through complementary base pairing.
Antibiotic resistance genes allow scientists to identify which cells have successfully taken up the recombinant plasmid.
When transformed bacteria are grown on agar containing the antibiotic, only those cells carrying the resistance gene will survive.
For example, a plasmid with an ampicillin resistance gene ensures that only transformed cells grow on ampicillin plates.
This selection step helps isolate recombinant bacteria for further analysis and gene expression studies.
When DNA fragments are inserted into vectors, they can enter in either orientation. To control directionality, scientists often use:
Two different restriction enzymes that produce non-identical sticky ends, ensuring the insert can only join one way.
Directional cloning using restriction sites positioned asymmetrically within the multiple cloning site.
This guarantees that the gene is in the correct orientation relative to promoter sequences for successful transcription and expression.
Electroporation efficiency—the proportion of cells successfully transformed—depends on several variables:
Voltage and pulse duration: Must be high enough to create temporary pores but not so strong that cells are damaged.
Cell type and membrane composition: Some bacterial species and mammalian cells are more resistant to pore formation.
DNA concentration and purity: Contaminants such as salts can cause arcing and reduce success rates.
Optimising these parameters ensures high transformation rates while maintaining cell viability.
Plasmids offer several advantages for gene cloning and expression:
They are small and easy to manipulate in the laboratory.
They replicate independently, producing multiple copies within each cell.
They can carry selectable markers and regulatory sequences that facilitate gene expression studies.
Bacteriophages are useful for large DNA fragments but require more complex handling and can be limited to certain bacterial hosts.
Practice Questions
Question 1 (2 marks)
Explain the role of restriction enzymes in the formation of recombinant DNA.
Mark scheme:
1 mark: Restriction enzymes cut DNA at specific recognition sites.
1 mark: This produces sticky or blunt ends that allow the target gene to be inserted into a vector with complementary ends.
Question 2 (5 marks)
Describe how a target gene can be inserted into a bacterial plasmid and introduced into a host cell during genetic engineering.
Mark scheme:
1 mark: Restriction enzymes cut both the target DNA and the plasmid at specific recognition sites.
1 mark: The cuts produce sticky ends on both DNA molecules that are complementary to each other.
1 mark: The target gene and plasmid are joined by DNA ligase, forming recombinant DNA.
1 mark: The recombinant plasmid is introduced into bacterial cells, for example, by electroporation or heat shock.
1 mark: Transformed cells are identified using selectable marker genes, such as antibiotic resistance or reporter genes.
